Determination of the Genome and Primary Transcriptome of Syngas Fermenting Eubacterium limosum ATCC 8486

Abstract

Autotrophic conversion of CO2 to value-added biochemicals has received considerable attention as a sustainable route to replace fossil fuels. Particularly, anaerobic acetogenic bacteria are naturally capable of reducing CO2 or CO to various metabolites. To fully utilize their biosynthetic potential, an understanding of acetogenesis-related genes and their regulatory elements is required. Here, we completed the genome sequence of the syngas fermenting Eubacterium limosum ATCC 8486 and determined its transcription start sites (TSS). We constructed a 4.4 Mb long circular genome with a GC content of 47.2% and 4,090 protein encoding genes. To understand the transcriptional and translational regulation, the primary transcriptome was augmented, identifying 1,458 TSSs containing a high pyrimidine (T/C) and purine nucleotide (A/G) content at the −1 and +1 position, respectively, along with 1,253 5′-untranslated regions, and principal promoter elements such as −10 (TATAAT) and −35 (TTGACA), and Shine-Dalgarno motifs (GGAGR). Further analysis revealed 93 non-coding RNAs, including one for potential transcriptional regulation of the hydrogenase complex via interaction with molybdenum or tungsten cofactors, which in turn controls formate dehydrogenase activity of the initial step of Wood-Ljungdahl pathway. Our results provide comprehensive genomic information for strain engineering to enhance the syngas fermenting capacity of acetogenic bacteria.

Functional cooperation of the glycine synthase-reductase and Wood–Ljungdahl pathways for autotrophic growth of Clostridium drakei

“…To confirm the functional role of a pathway, a reconstruction of C. drakei to modify the pathways is required to confirm the prior result, but, unfortunately, developing a genetic modification tool for the strain was infeasible. As an alternative, a genetically modifiable acetogen, the Eubacterium limosum ATCC 8486 strain with the absence of GSRP-coding genes in its genome, was examined for the heterogeneous introduction of the pathway (8,31). To confirm the functional role, the GSRP-encoding gene cluster from the C. drakei genome was cloned into a plasmid, which was then introduced into E. limosum (the GSRP strain), and the same plasmid backbone without the gene cluster was introduced into E. limosum as a control strain (SI Appendix, Fig.…”

“…With this advantage, acetogens are considered to be the most promising industrial platform to produce biofuels and chemical commodities through synthesis gas fermentation (1)(2)(3)(4). Although gene composition and arrangement of the WLP vary among acetogens, the WLP-coding genes are well-conserved, along with two genes encoding a partial glycine synthase: the glycine cleavage system H protein (gcvH) and dihydrolipoyl dehydrogenase (lpdA) genes (5)(6)(7)(8). The glycine synthase pathway was initially proposed for the utilization of CO 2 under autotrophic growth conditions (2).…”

A TetR-family protein activates transcription from a new promoter motif associated with essential genes for autotrophic growth in acetogens

“…We next investigated if P cauto was represented in other industrially relevant acetogens with available genomes: Clostridium ljungdahlii , C. ragsdalei , C. coskatii , Moorella thermoacetica , and Eubacterium limosum (Bengelsdorf et al, 2016; Redl et al, 2017; Shin et al, 2016; Song et al, 2017). We performed the reverse of the methodology previously used to search for consensus sequence motifs by looking for the occurrence of P cauto 300 nt upstream of annotated genes (since no TSS data was available) using the FIMO tool (Grant et al, 2011) within MEME.…”

“…TSSs and promoter motifs) and a more accurate annotation of acetogen genomes have the potential to yield valuable insights into the complex transcriptional regulation of acetogens. To date, only one study has determined TSSs in acetogens, using Eubacterium limosum (Song et al, 2017). Here, we used dRNA-Seq as a tool to identify the TSSs in the model-acetogen Clostridium autoethanogenum grown under autotrophic and heterotrophic conditions.…”

Adaptive Laboratory Evolution of Eubacterium limosum ATCC 8486 on Carbon Monoxide

“…, at a pressure of 200 kPa and 50 mL of headspace filled with 0%, 20%, 40%, 60%, 80%, and 100% CO that is balanced using 100%, 80%, 60%, 40%, 20%, and 0% N 2 , respectively, for autotrophic growth conditions. To enhance the autotrophic growth rate during adaptation, 40 mM NaCl was supplemented to the media, which couples with sodium dependent ATP synthase (Jeong et al, 2015;Song et al, 2017).…”